CRISPR/Cas9 and genome editing for genetic neuromuscular disorders

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1 CRISPR/Cas9 and genome editing for genetic neuromuscular disorders Annemieke Aartsma-Rus AFDELING HUMANE GENETICS, LUMC, LEIDEN

2 Disclosures Employed by LUMC, which has patents on exon skipping technology, some of which has been licensed to BioMarin and subsequently sublicensed to Sarepta. As co-inventor of some of these patents I am entitled to a share of royalties Ad hoc consultant for PTC Therapeutics, BioMarin Pharmaceuticals Inc., Alpha Anomeric, Global Guidepoint, GLG consultancy, Grunenthal, Wave, Sarepta, Eisai and BioClinica Member of the scientific advisory boards of ProQR, MirrX therapeutics and Philae Pharmaceuticals. Remuneration for these activities paid to LUMC. LUMC received speaker honoraria from PTC Therapeutics and BioMarin Pharmaceuticals. Sept 2018

3 Muscular dystrophies shared pathology Connection cytoskeleton muscle fibers and extracellular matrix is lost/impaired due to genetic mutations Less stability during muscle contraction Musclefibers more pronetodamage Pathology: Continuous damage of muscle fibers Chronic inflammation Fibrosis/adiposis and suppressed regeneration Continuous loss of muscle tissue and function 3 Aartsma-Rus

4 Muscular dystrophies Treatment often is multidisciplinary care Research ongoing to restore missing protein with genetic therapies For Duchenne some approved drugs Ataluren(EMA) and eteplirsen(fda) Mutation specific Not available everywhere in Europe Not applicable to all patients Genomeediting tothe rescue 4 Aartsma-Rus

5 Genome editing for Duchenne Use DMD as showcase What is CRISPR/Cas9 and how does it work? How can it be used as a therapy for DMD? Why is everyone so enthusiastic What has been done? What still needs to be done? Misconceptions 5 Aartsma-Rus

6 Genome editing CRISPR system ( guide RNA ) Cas9 6 Aartsma-Rus

7 Why would you cut DNA? DNA contains genes (genetic blueprint) DNA damage is NOT good Our DNA is damaged continuously This is repaired ASAP 7 Aartsma-Rus

8 DNA damage repair DNA damage Repair Recombination (dividing cells) Errorless repair NHEJ (non dividing cells) Non homologous end joining Information is lost 8 Aartsma-Rus

9 DNA damage repair in muscle Muscles and neurons are postmitotic cells For NMDs only the NHEJ system available This does not correct mutations, but generates mistakes in the DNA Why would you want this? Permanent exon skipping Recap: what is exon skipping? 9 Aartsma-Rus

10 Gene to protein Exons Introns 7 Gene (DNA) Splicing messenger RNA RNA copy (pre mrna) dystrophin protein 10 Aartsma-Rus

11 Duchenne: genetic code disrupted 11 Aartsma-Rus

12 Becker: genetic code maintained 12 Aartsma-Rus

13 Restore genetic code AON Exon 47 Intron 47/50 Exon 51 Intron 51 Exon 52 Intron 52 Code repaired Exon 46 Exon 47 Exon 52 Partially functional dystrophin 13 Aartsma-Rus

14 Exon skipping Repairs code on transcript (RNA) level Temporary effect: weekly treatment Repair code on DNA level permanent effect This can be done with genome editing Advantages over exon skipping Single treatment Can also skip multiple exons Works for duplications 14 Aartsma-Rus

15 It works! In mice. 15 Aartsma-Rus

16 What did they do in mice? 16 Aartsma-Rus

17 What did they do in mice? Local delivery DNA and RNA analysis Local delivery Protein analysis Nelson et al., Science 2016; 351: Aartsma-Rus

18 What did they do in mice? Systemic delivery Protein analysis Long et al., Science 2016; 351: Aartsma-Rus

19 So what did they achieve in mice Generated the target deletion or skip on DNA level Restored dystrophin BUT Efficiency rather low Systemic delivery challenging Need viral vector to deliver CAS9 and guidernas 19 Aartsma-Rus

20 Tests in patient-derived cells 20 Aartsma-Rus

21 Multiexon skipping 21 Aartsma-Rus

22 What did they do in patient cells? Multiexon skipping Oosterhout et al., Nat Comm2015; 6: Aartsma-Rus

23 Tests in patient derived cells Exon skippen Genome editing 23 Aartsma-Rus

24 What did they do in patient cells? Duplication mutation (exon duplication) Wojtalet al., AJHG 2016; 98: Aartsma-Rus

25 Why are people so enthusiastic? Technique has potential Targeted modification of DNA Offers opportunities not available before Generating disease models (cells and animals) Therapeutic options Easy (in cell cultures) Many examples of it working in DMD models Media attention Recent paper in dogs in science: showed dystrophin restoration after systemic treatment in 1 dog 25 Aartsma-Rus

26 Media hype 26 Aartsma-Rus

27 What still needs to be solved? Delivery Need to deliver Cas9 and guide RNAs to all muscles Viral vectors (AAV) Tested for gene addition with promising results Here: two-component system and two-step process Manufacturing. Efficiency Currently very low Safety How specific are the Cas9 enzymes? Integration of AAV vectors 27 Aartsma-Rus

28 CRISPR Cure For most NMDs: restoring code will not result in restoring functional protein For DMD genetic code is restored Becker like proteins, partially functional This is not a cure This does not halt muscular dystrophy Effect depends on time of intervention Loss of muscle and muscle function is irreversible So need to treat early 28 Aartsma-Rus

29 Genome editing of embryos You need to know that parents are carriers (recessive) or one parent is a carrier (dominant/x-recessive BUT.if you know this, embryo selection is a less risky and less invasive option 29 Aartsma-Rus

30 Summary CRISPR/Cas9 enables editing of the genome Potential for generating model systems There is therapeutic potential, BUT For muscle diseases delivery is very challenging as yet Outstanding questions on safety If it works, it will slow down disease progression ( CRISPR Cure is a misnomer) 30 Aartsma-Rus